This application addresses PA-11-260 Research Project Grant (Parent R01). Chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL1-negative) chronic myeloid leukemia (aCML) are both hematologic malignancies that have historically been diagnosed based on neoplastic expansion of granulocytic cells and exclusion of genetic drivers known to occur in other myeloproliferative neoplasms (MPN). The absence of defining genetic lesions has made diagnosis of these diseases challenging, and resulted in a dearth of effective therapeutic options for patients. I have recently identified gain-of-function CSF3R mutations in neoplastic cells from ~60% of CNL and aCML patients(1). These CSF3R mutations induce activation of downstream kinase signaling pathways resulting in hypersensitivity of CSF3R-mutant cells to FDA-approved small-molecule kinase inhibitors. Several patients harboring CSF3R mutations have been treated with these kinase inhibitors as single-agents and have exhibited dramatic and durable clinical responses. These findings lead to a number of important new questions and project directions. My long-term goal is to establish CSF3R targeted therapies as a pillar of effective long-term disease management for CNL/aCML patients. My immediate goals are to comprehensively understand the molecular mechanisms by which CSF3R drives leukemogenesis, to define the combinatorial therapeutic regimens that can control CSF3R- driven disease, and to identify alternative drivers in cases without CSF3R mutation. Based on the central hypothesis that CSF3R and related pathways are onco-requisite for the pathogenesis of CNL and aCML, I predict that targeting these pathways will revolutionize clinical care and outcomes for CNL/aCML patients. To accomplish these goals, several specific questions will be addressed: 1) What are the molecular mechanisms by which CSF3R mutation leads to receptor activation? CSF3R mutations fall into two categories based on spatial location within the protein, and preliminary data indicate these two classes of CSF3R mutations exhibit distinct mechanisms of activation and drug sensitivity patterns. It will be important to fully elucidate the molecular mechanisms underlying these phenotypes. 2) What are the consequences of combinatorial mutations involving CSF3R? My recently published data indicate that a substantial proportion of CSF3R mutant cases also harbor secondary mutations within the same allele of CSF3R or within secondary genes such as SETBP1. I will examine the effects on signaling and drug sensitivity of these combinatorial mutations in the context of single-agent and combination therapeutic regimens, both in vitro and in vivo. 3) What are the genetic drivers in CNL/aCML cases without CSF3R mutation? I have performed deep sequencing on CNL/aCML cases without CSF3R mutation, and have identified candidate mutations within the CSF3R pathway or related pathways in each case. I will validate the transformative capacity and drug sensitivity of each of these candidate driver oncogenes. Cumulatively, I expect these innovative analyses to have a major impact on our understanding of CNL/aCML biology and successful clinical management of these diseases.